Tocochromanols are recognized for nutritional content, plant stress response, and seed longevity. Here we present a systems biological approach to characterize and develop predictive assays for genes affecting tocochromanol variation in barley. Major QTL, detected in three regions of a SNP linkage map, affected multiple tocochromanol forms. Candidate genes were identified through barley/rice orthology and sequenced in genotypes with disparate tocochromanol profiles. Gene-specific markers, designed based on observed polymorphism, mapped to the originating QTL, increasing R2 values at the respective loci. Polymorphism within promoter regions corresponded to motifs known to influence gene expression. Quantitative PCR analysis revealed a trend of increased expression in tissues grown at cold temperatures. These results demonstrate utility of a novel method for rapid gene identification and characterization, and provide a resource for efficient development of barley lines with improved tocochromanol profiles.